This research was supported by the Intramural Research Program of the NIH, NCI, and CCR. All authors received Intramural Research Grant from NIH, NCI, and CCR.

1. Prepare Reagents
<ol>
	<li>1x PBS
	<ol>
		<li>Dilute 100 mL 10x PBS with 900 mL dH2O and adjust the pH to 7.4 using a pH meter. Store at room temperature.</li>
	</ol>
	</li>
	<li>Lysis solution (LS)
	<ol>
		<li>Prepare 2.5 M NaCl, 100 mM disodium EDTA, 10 mM Tris base, and 200 mM NaOH in 900 mL dH2O; it commonly takes about 20 min to allow the mixture to fully dissolve. Adjust the pH to 10 using a pH meter. Add 1% sodium lauryl sarcosinate and 1% Triton X-100, and adjust the final volume to 1,000 mL. Cool to 4 °C for at least 30 min before use.</li>
	</ol>
	</li>
	<li>Alkaline electrophoresis solution (AES), pH &gt;13
	<ol>
		<li>Prepare 200 mM NaOH and 1 mM disodium EDTA in 800 mL dH2O. Adjust the pH and make sure that it is pH &gt;13. Adjust the final volume to 1,000 mL. Make fresh before use and cool to 4 °C for at least 30 min before use.</li>
	</ol>
	</li>
	<li>Neutral electrophoresis sollution (NES)
	<ol>
		<li>Prepare 1,000 mL neutral electrophoresis buffer by mixing 100 mM Tris base and 300 mM sodium acetate to 1,000 mL dH2O. Adjust the pH to 9.0 with glacial acetic acid. Cool to 4 °C for at least 30 min before use.</li>
	</ol>
	</li>
	<li>DNA precipitation solution (DPS)
	<ol>
		<li>Preparation of 10 mL 7.5 M ammonium acetate stock. For 50 mL of DNA precipitation solution, mix 6.7 mL 7.5 M ammonium acetate with 43.3 mL 95% ethanol. Store at room temperature.</li>
	</ol>
	</li>
	<li>Staining solution
	<ol>
		<li>Add 1 µL 10,000x green fluorescent nucleic acid stain (e.g., SYBR Green) in 30 mL Tris-EDTA buffer (10 mM Tris-HCl, 1 mM disodium EDTA, pH 7.4) and store at 4 °C. Protect from light.</li>
	</ol>
	</li>
	<li>1% low melting agarose
	<ol>
		<li>Melt 1% low melting point agarose (1 g in 100 mL dH2O) in a microwave. Swirl the agarose every 15-20 s to make sure that the agarose is completely molten. Place the agarose in 37 °C water bath for at least 20 min before use.</li>
	</ol>
	</li>
	<li>Pre-warm pipette tips
	<ol>
		<li>Cut off the narrow ends of P200 pipette tips by 3 mm and warm at 37 °C before pipetting agarose.</li>
	</ol>
	</li>
</ol>
2. Prepare Comet Slides
<ol>
	<li>Slide coating
	<ol>
		<li>Melt 1% agarose (1 g in 100 mL dH2O) in a microwave for 2 - 3 min or until the agarose is completely molten. Dip the glass microscope slides into the agarose and wipe one side of the slide using a lint-free wipe.</li>
		<li>Lay the slides on a flat surface to air-dry or heat at 50 °C for faster drying; a transparent agarose film should be formed after drying. Place the coated slides in 37 °C before use.</li>
	</ol>
	</li>
	<li>Preparation of single cell suspensions
	<ol>
		<li>Culture and treat the glioma cell
		<ol>
			<li>Culture the U251 MG cells in DMEM-Ham F-12 medium supplemented with 10% FBS, 100 U/mL penicillin, and 10 µg/mL streptomycin at 37 °C with 5% CO2.</li>
			<li>Digest the cells using 1 mL trypsin for 3 min, and neutralize trypsin using DMEM-Ham F-12 medium with FBS. Collect in 15 mL tube, spin at 300 x g for 4 min, aspirate the medium, and suspend cells at 2 x 105 cell/mL in 1x PBS. 
			NOTE: The cell sample should be prepared immediately before starting the assay and all samples should be handled in a dark or dimmed environment to prevent DNA damage from light.</li>
			<li>Combine the cell suspension with 1% molten low melting point agarose (at 37 °C) at a ratio 1:10 (v/v), mix gently by pipetting up and down, and immediately pipette 30 µL onto a slide. Use the side of the the pipette tip to spread the agarose/cell mixture to ensure the formation of a thin layer.</li>
			<li>Place the slide flat at 4 °C in the dark for 10 min. Increasing the gelling time to 30 min improves adherence of samples in high humidity environments.</li>
			<li>Immerse the slide in 4 °C LS in the dark for 1 h to overnight.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Single Cell Electrophoresis
<ol>
	<li>Proceed to alkaline (step 3.2) or neutral (step 3.3) comet assay</li>
	<li>For alkaline comet assay
	<ol>
		<li>Gently remove slides from the LS, drain excess buffer, and gently immerse in AES for 1 h at 4 °C to allow DNA unwinding. Keep the slides in the dark.</li>
		<li>Add pre-chilled AES in the electrophoresis slide tray, do not exceed 0.5 cm above the slides (this depends on the size of the electrophoresis units), place the slides inside and cover with a cap. Set the power supply voltage to 1 V/cm (the length between electrodes) and run for 30 min at 4 °C.</li>
		<li>Drain excess electrophoresis solution from slide. Gently immerse slides twice in dH2O for 5 min each at room temperature.</li>
		<li>Gently immerse slides in 70% ethanol for 5 min at room temperature. Proceed to step 4.</li>
	</ol>
	</li>
	<li>For neutral comet assay
	<ol>
		<li>Gently remove the slides from the LS, drain excess buffer, and gently immerse in NES for 30 min at 4 °C. Keep the slide in the dark.</li>
		<li>Add pre-chilled neutral electrophoresis buffer in the electrophoresis slide tray, do not exceed 0.5 cm above slides (this depends on the size of the electrophoresis units), place the slides inside and cover with a cap. Set the power supply voltage to 1 V/cm (the length between electrodes) and run for 45 min at 4 °C.</li>
		<li>Drain excess buffer from the slides. Gently immerse slides in DPS for 30 min at room temperature.</li>
		<li>Gently immerse the slides in 70% ethanol for 30 min at room temperature. Proceed to step 4.</li>
	</ol>
	</li>
</ol>
4. Stain Comet Slides
<ol>
	<li>Dry slides at 37 °C for 10 - 15 min in the dark.</li>
	<li>Place 50 - 100 µL green fluorescent nucleic acid staining solution onto each dried agarose and stain for 15 min at room temperature in the dark.</li>
	<li>Rinse the slides briefly in dH2O and dry completely at 37 °C in the dark. Proceed to image acquisition and analysis.</li>
</ol>
5. Image Acquisition and Analysis
NOTE: The visualization and quantification of DNA breaks are based on epifluorescence microscopy and the comet assay software (see Table of Materials)12.
<ol>
	<li>Place the slides on the microscope with a slide holder. Ensure the agarose gel is facing to the objective lens. Randomly capture images from the stained comet slides using a fluorescence microscope with a 10x objective lens. Avoid the edges and the areas around any air bubbles.</li>
	<li>Ensure each comet tail is horizontally distributed. Comet heads should originate from the left and the tail from the right.</li>
	<li>Save each picture in a binary TIF format with bright DNA stain and dark background. Load images to the software using the "Select files to analyze" button, which is located on the left of the toolbar. An image view window should appear (Figure 1).</li>
	<li>Draw a measurement frame on the screen and adjust its size in accordance with the comet of the cell. Click the "Adjust" button to set up the threshold of the head, comet, and tail according to the image, then click the "Start measurements" button (Figure 1).</li>
	<li>Select a cell using the frame and activate the measurement by clicking with the mouse on the "Assay the comet" button; an intensity image shows up on the "profiles" window with the selected measurement parameters. The results can be saved by clicking the "store result" button (Figure 1). 
	NOTE: The software calculates the parameters including the length of the comet tail, the percentage of the tailed DNA, the tail moment (TM) and the Olive tail moment (OTM). The tail moments are calculated by the formulas as follow: 
	<img alt="TM calculation formula: tail DNA analysis in genomics, equation for tail length and percentage." src="/files/ftp_upload/56450/56450eq1.jpg" data-altupdatedat="1747911167000" data-alt="Equation 1"> 
	<img alt="OTM equation for DNA analysis; formula: (Tail mean - Head mean)/100 × Tail%DNA." src="/files/ftp_upload/56450/56450eq2.jpg" data-altupdatedat="1747911167000" data-alt="Equation 2"></li>
	<li>Analyze at least 50 cells per treatment.</li>
</ol>

<ol>
	<li>Ostling, O., Johanson, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microelectrophoretic+study+of+radiation-induced+DNA+damages+in+individual+mammalian+cells.">Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells.</a> Biochem Biophys Res Commun. 123 (1), 291-298 (1984).</li><li>Singh, N. P., McCoy, M. T., Tice, R. R., Schneider, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+technique+for+quantitation+of+low+levels+of+DNA+damage+in+individual+cells.">A simple technique for quantitation of low levels of DNA damage in individual cells.</a> Exp Cell Res. 175 (1), 184-191 (1988).</li><li>Tice, R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+cell+gel/comet+assay:+guidelines+for+in+vitro+and+in+vivo+genetic+toxicology+testing.">Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.</a> Environ Mol Mutagen. 35 (3), 206-221 (2000).</li><li>Shah, A. J., Lakkad, B. C., Rao, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotoxicity+in+lead+treated+human+lymphocytes+evaluated+by+micronucleus+and+comet+assays.">Genotoxicity in lead treated human lymphocytes evaluated by micronucleus and comet assays.</a> Indian J Exp Biol. 54 (8), 502-508 (2016).</li><li>Azqueta, A., Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essential+comet+assay:+a+comprehensive+guide+to+measuring+DNA+damage+and+repair.">The essential comet assay: a comprehensive guide to measuring DNA damage and repair.</a> Arch Toxicol. 87 (6), 949-968 (2013).</li><li>Goldstein, M., Kastan, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+damage+response:+implications+for+tumor+responses+to+radiation+and+chemotherapy.">The DNA damage response: implications for tumor responses to radiation and chemotherapy.</a> Annu Rev Med. 66, 129-143 (2015).</li><li>Gavande, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+targeted+therapy:+The+past+or+future+of+cancer+treatment?.">DNA repair targeted therapy: The past or future of cancer treatment?.</a> Pharmacol Ther. 160, 65-83 (2016).</li><li>Torgovnick, A., Schumacher, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+repair+mechanisms+in+cancer+development+and+therapy.">DNA repair mechanisms in cancer development and therapy.</a> Front Genet. 6, 157 (2015).</li><li>Weston, V. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PARP+inhibitor+olaparib+induces+significant+killing+of+ATM-deficient+lymphoid+tumor+cells+in+vitro+and+in+vivo.">The PARP inhibitor olaparib induces significant killing of ATM-deficient lymphoid tumor cells in vitro and in vivo.</a> Blood. 116 (22), 4578-4587 (2010).</li><li>Brown, J. S., O'Carrigan, B., Jackson, S. P., Yap, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+DNA+Repair+in+Cancer:+Beyond+PARP+Inhibitors.">Targeting DNA Repair in Cancer: Beyond PARP Inhibitors.</a> Cancer Discov. 7 (1), 20-37 (2017).</li><li>Lu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemosensitivity+of+IDH1-Mutated+Gliomas+Due+to+an+Impairment+in+PARP1-Mediated+DNA+Repair.">Chemosensitivity of IDH1-Mutated Gliomas Due to an Impairment in PARP1-Mediated DNA Repair.</a> Cancer Res. 77 (7), 1709-1718 (2017).</li><li>Konca, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+cross-platform+public+domain+PC+image-analysis+program+for+the+comet+assay.">A cross-platform public domain PC image-analysis program for the comet assay.</a> Mutat Res. 534 (1-2), 15-20 (2003).</li><li>Valverde, M., Rojas, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+and+occupational+biomonitoring+using+the+Comet+assay.">Environmental and occupational biomonitoring using the Comet assay.</a> Mutat Res. 681 (1), 93-109 (2009).</li><li>Collins, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comet+assay+for+DNA+damage+and+repair:+principles,+applications,+and+limitations.">The comet assay for DNA damage and repair: principles, applications, and limitations.</a> Mol Biotechnol. 26 (3), 249-261 (2004).</li><li>Karbaschi, M., Cooke, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+method+for+the+high-throughput+processing+of+slides+for+the+comet+assay.">Novel method for the high-throughput processing of slides for the comet assay.</a> Sci Rep. 4, 7200 (2014).</li></ol>

The authors have nothing to disclose.

The comet assay is an efficient tool to measure single and double-strand DNA breaks at the cellular level. The assay has been widely applied as a "golden standard" in studies regarding genotoxicity and biomonitoring13, ranging from base lesions, DNA crosslinks, drug development, and alkali sensitive sites. In the present study, we showed two distinct step-by-step protocols for alkaline and neutral comet assays, respectively. Combining single cell electrophoresis, fluorescent microscopy, and image interpretation, both the alkaline and neutral comet assays provide quantitative approaches to evaluate the extent of DNA damage in vitro.
Several key steps are essential for the successful execution of the comet assay. For example, care should be taken in the steps of reagent preparation. Each solution should be freshly prepared and kept at the appropriate temperature for at least 30 min before use, to prevent endogenous DNA damage or repair during sample preparation. Another vital step is the preparation of viable single-cell suspension. It is important to keep the agarose at 37 &#176;C before use, to maximize cell survival during preparation of the comet slides. The cell/agarose suspension should be prepared immediately before starting the assay. Using the side of pipette tips to spread the cell/agarose suspension helps to achieve a thin layer of agarose on the slides, so that most of the cells can be positioned in the same focal plane during microscopy. Optionally, scratch the edges of the slides using a diamond-tipped pen to improve the agarose attachment. 
Despite the advantages of the comet assay, there are several limitations of this traditional method14,15. For example, the throughput of the comet assay is limited by the process of the glass slides preparation. In some occasions, an eight-welled glass slide provides a solution to improve assay throughput. Another limitation is that the comet assay simply represents the ratio of the fragmented DNA in cells. Additional supportive information is required to thoroughly depict the cellular changes besides DNA damage. In that case, apoptosis analysis, &#947;H2A.X staining, and immunoblotting could be very helpful to provide a thorough analysis of different aspects to reveal the effect of DNA damaging agents. 
There are numerous software for comet image analysis and the output includes a variety of different parameters. The most commonly used parameters are the tail length, the percentage of DNA in the tail, and the tail moment. Tail length only can be used at low levels of DNA damage, since it does not tend to change once the tail is established14. Subsequently, the intensity of the tail increases as the damage is enhanced. The percentage of DNA in the comet tail is another useful parameter, as it is linearly related to the breaking frequency. The tail moment combines tail length and tail intensity in one single value, thus, it is the most useful and frequently used parameter. 
The comet assay has been widely applied in genotoxicity testing and human biomonitoring. With the development of DNA repair enzyme inhibitors, the comet assay could a valuable tool for testing combination therapies that improve the outcome of traditional chemotherapy.

The comet assay was first developed by Ostling and Johanson in 1984 by demonstrating the migration of DNA fragments from nuclei under a neutral condition1. The technique was later developed by Singh et al., showing that an alkaline condition substantially increased the specificity and reproducibility of the assay2. Since then, the neutral comet assay is mostly used to detect double-stranded DNA breaks, whereas the alkaline comet assay is more sensitive for smaller amounts of DNA damage, including single and double strand DNA breaks, alkali-labile sites, DNA-DNA or DNA-protein cross-linking, and DNA single-strand breaks associated with incomplete excision repair sites3,4. Both assays allow visualization of fragmented DNA, and provide a straightforward way to quantitatively evaluate DNA damage. The comet assay is regarded as a sensitive method for in vitro and in vivo genetic toxicological studies and is applicable to different research areas, such as early drug-candidate selection, environmental monitoring, human biomonitoring, and fundamental research in DNA damage and repair5. 
The principle of the assay is that under an electric field, fragmented DNA migrates out of the nucleoid body (also known as the "comet head") and forms a DNA stain in the agarose gel (also known as the "comet tail"). With nucleotide staining, the extent of DNA damage can be quantified by analyzing "comets" formed by this single cell electrophoresis. Calculation of the tail moment can further help to compare DNA damage among different experimental groups. Compared with traditional methods of DNA damage detection, the comet assay is direct, sensitive, inexpensive, and relatively simple.
Radiotherapy and chemotherapies are common strategies for cancer treatment by generating single strand and double strand DNA breaks in chromosomes6. The recent advancement in DNA repair inhibitors allows a more effective genotoxic effect by combination chemotherapy, and therefore, potentially reduces systemic side effects such as anemia, infection, and bone marrow suppression7,8. In this study, we showed the investigation of a poly (ADP-ribose) polymerase (PARP) inhibitor, olaparib (Ola)9. PARP is an abundant nuclear protein and is responsible for DNA base excision repair by forming a poly (ADP-ribose) polymer10. Temozolomide (TMZ) is an orally available alkylating agent and has been widely used for glioma patient treatment. By using the comet assay to quantify DNA damage, we demonstrate that combining olaparib with temozolomide profoundly enhances the DNA damage in glioma cells, which suggests olaparib/temozolomide combination therapy is an effective strategy to treat glioma, as compared with temozolomide alone11.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10x PBS(Ca&lt;sup&gt;++&lt;/sup&gt;, Mg&lt;sup&gt;++&lt;/sup&gt; free)</td><td>TEKnova</td><td>P0196</td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>S5886</td><td></td></tr><tr><td>EDTA</td><td>TEKnova</td><td>E0308</td><td></td></tr><tr><td>Trizma base</td><td>Sigma</td><td>T1503</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>72068</td><td></td></tr><tr><td>Sodium lauryl sarcosinate</td><td>Sigma</td><td>L7414</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>93443</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma</td><td>32318</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma</td><td>695092</td><td></td></tr><tr><td>Ammonium acetate</td><td>Sigma</td><td>A1542</td><td></td></tr><tr><td>SYBR Green</td><td>Invitrogen</td><td>S33102</td><td></td></tr><tr><td>Low melting point agarose</td><td>Invitrogen</td><td>16520</td><td></td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500</td><td></td></tr><tr><td>95% ethanol</td><td>WARNER-GRAHAM</td><td>#64-17-5</td><td></td></tr><tr><td>Trypsin</td><td>GIBICO</td><td>25300-054</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Glass tissue slides</td><td>ELECTRON MICROSCOPY SCIENCES</td><td>63422-11</td><td></td></tr><tr><td>Kimwipes</td><td>KIMberly-Clark</td><td></td><td></td></tr><tr><td>1.5 mL Microcentrifuge Tubes</td><td>DENVILLE</td><td></td><td></td></tr><tr><td>Pipette Tips</td><td>SHARP</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microwave</td><td>Avanti</td><td></td><td></td></tr><tr><td>Waterbath</td><td>PRECISION</td><td></td><td></td></tr><tr><td>Horizontal electrophoresis chamber</td><td>TREVIGEN</td><td>Cometassay ES II</td><td></td></tr><tr><td>Power supply</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Incubator</td><td>Quincy Lab</td><td>Model 12-140E</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Zeiss</td><td>LSM700</td><td></td></tr><tr><td>Micropipettor</td><td>Eppendorf</td><td></td><td></td></tr></tbody></table>

evaluating in vitro dna damage using comet assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

The present protocol describes a step-by-step workflow for the comet assay execution and data analysis (Figure 1). Results from the alkaline and neutral comet assays showed that the comet tail of doxorubicin-treated U251 cells (1 µM, 20 h) was longer and had higher DNA intensity, suggesting a substantial accumulation of fragmented DNA due to chemotherapy (Figure 2).
Quantitative analysis for either the alkaline or neutral comet assay showed significantly increased comet tail formation after doxorubicin treatment (Figure 3A) indicating DNA damage. Results are presented as mean ± SEM, and t-tests were used for statistical comparisons. Quantification of the tail moment from both assays confirmed the increase of average tail moment: For the alkaline comet assay, 0.134 ± 0.0448 (n = 52) vs. 13.84 ± 1.325 (n = 51), p &lt;0.01; For the neutral comet assay, 0.596 ± 0.06668 (n = 78) vs. 9.979 ± 0.658 (n = 51), p &lt;0.01. These data provide a quantifiable index to evaluate DNA damage. Moreover, combination treatment of PARP inhibitor olaparib and temozolomide significantly enhanced DNA damage in glioma cells (Figure 3C), with an increased length and intensity of the comet tail signal. Olaparib alone did not introduce DNA damage, whereas it potentiated the genotoxic effect of the chemo agent temozolomide (DMSO, 0.9836 ± 0.3377 (n = 64); Olaparib, 0.6663 ± 0.2325 (n = 51); TMZ, 6.197 ± 0.572 (n = 51); Olaparib + TMZ, 44.04 ± 1.269(n = 115), p &lt;0.01).
<img alt="Alkaline comet assay analysis; DNA damage; image, intensity profiles; measurement frame, software tools." class="xfigimg" src="/files/ftp_upload/56450/56450fig1.jpg" data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1: Step-by-step guide of image analysis using comet assay software. (A) The toolbar of the comet assay software. (B) Alkaline comet assay as an example: the user interface of comet assay software consists of an image view, intensity profile, and data acquisition window. (C) The image background, comet region, and comet head were defined in the software. (D) The intensity profiles of the comet head (red) and tail (green) are plotted, respectively. <a href="https://cloudflare.jove.com/files/ftp_upload/56450/56450fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="comet assay DNA damage analysis, alkaline and neutral, DMSO vs Dox treated, fluorescence image" class="xfigimg" src="/files/ftp_upload/56450/56450fig2.jpg" data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2: Representative images of the alkaline and neutral comet assays. U251 glioma cells were treated with doxorubicin and investigated by alkaline (upper panels) and neutral (lower panels) comet assays (Scale bar = 10 µm). <a href="https://cloudflare.jove.com/files/ftp_upload/56450/56450fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Alkaline and neutral comet assay results, DNA damage comparison, charts, fluorescent signals." class="xfigimg" src="/files/ftp_upload/56450/56450fig3.jpg" data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3: Representative data of the comet tail moment. (A) Alkaline comet assay data plot. **p &lt;0.01. (B) Neutral comet assay data plot. **p &lt;0.01. (C) Comet assay analysis revealed the synergistic effect of combination therapy including temozolomide (TMZ) and olaparib (Ola). **p &lt;0.01. (D) Representative images of glioma cells under combination treatment of temozolomide and olaparib (Scale bar = 10 µm). <a href="https://cloudflare.jove.com/files/ftp_upload/56450/56450fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Evaluating In Vitro DNA Damage Using Comet Assay at JoVE.com

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

37.3K Views.  National Cancer Institute. The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

Video: Evaluating In Vitro DNA Damage Using Comet Assay

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. Traditional 3D assays such as the Boyden Chamber require that cells are displaced from the original culture location and moved to a new environment. Not only does this disrupt the cellular processes that are intrinsic to the microenvironment, but it often results in a loss of cells. These problems are especially challenging when dealing with cells that are either rare, or extremely sensitive to their microenvironment. Here, we describe the development of a 3D invasion assay that avoids both concerns. In this assay, cells are plated within a small well and an ECM matrix containing a chemoattractant is laid atop the cells. This requires no cell displacement, and allows the cells to invade upwards into the matrix. In this assay, cell invasion as well as cell morphology can be assessed within the collagen gel. Using this assay, we characterize the invasive capacity of rare and sensitive cells; the hybrid cells resulting from fusion between breast cancer cells MCF7 and mesenchymal/multipotent stem/stroma cells (MSCs).

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

This protocol describes an inverted vertical invasion assay that could be used to quantify the migration and invasion capabilities of cells in a three-dimensional setting while preserving the cell microenvironment. This assay is suitable for rare and/or sensitive cells.

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human central nervous system (CNS). However, both the cytological origin and the evolutionary process of HBs (including neovascularization) remain controversial, and anti-angiogenesis for VHL-HBs, based on classic HB angiogenesis, have produced disappointing results in clinical trials. One major obstacle to the successful clinical translation of anti-vascular treatment is the lack of a thorough understanding of neovascularization in this vascular tumor. In this article, we present a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in HBs, as well as its role in HBs. With this procedure, researchers can accurately understand the complexity of HB neovascularization and identify the function of this common form of angiogenesis in HBs. These protocols can be used to evaluate the most promising anti-vascular therapy for tumors, which has high translational potential either for tumors treatment or for aiding in the optimization of the anti-angiogenic treatment for HBs in future translations. The results highlight the complexity of HB neovascularization and suggest that this common form angiogenesis is only a complementary mechanism in HB neovascularization.

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

This paper presents a comprehensive procedure to evaluate in vitro whether classic tumor angiogenesis exists in hemangioblastomas (HBs) and its role in HBs. The results highlight the complexity of HB-neovascularization and suggest that this common form of angiogenesis is only a complementary mechanism in the HB-neovascularization.

The inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene plays a crucial role in the development of hemangioblastomas (HBs) within the human ...

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Bioengineering

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other environmental stressors. Use of the comet assay in regulatory testing for genotoxic potential in rodents has been driven by adoption of an Organisation for Economic Co-operation and Development (OECD) test guideline in 2014. Comet assay slides are typically prepared from fresh tissue at the time of necropsy; however, freezing tissue samples can avoid logistical challenges associated with simultaneous preparation of slides from multiple organs per animal and from many animals per study. Freezing also enables shipping samples from the exposure facility to a different laboratory for analysis, and storage of frozen tissue facilitates deferring a decision to generate DNA damage data for a given organ. The alkaline comet assay is useful for detecting exposure-related DNA double- and single-strand breaks, alkali-labile lesions, and strand breaks associated with incomplete DNA excision repair. However, DNA damage can also result from mechanical shearing or improper sample processing procedures, confounding the results of the assay. Reproducibility in collection and processing of tissue samples during necropsies may be difficult to control due to fluctuating laboratory personnel with varying levels of experience in harvesting tissues for the comet assay. Enhancing consistency through refresher training or deployment of mobile units staffed with experienced laboratory personnel is costly and may not always be feasible. To optimize consistent generation of high quality samples for comet assay analysis, a method for homogenizing flash frozen cubes of tissue using a customized tissue mincing device was evaluated. Samples prepared for the comet assay by this method compared favorably in quality to fresh and frozen tissue samples prepared by mincing during necropsy. Moreover, low baseline DNA damage was measured in cells from frozen cubes of tissue following prolonged storage.

This protocol describes several procedures for preparing high quality frozen tissue samples at the time of necropsy for use in the comet assay to assess DNA damage: 1) minced tissue, 2) scraped epithelial cells from the gastrointestinal tract, and 3) cubed tissue samples, requiring homogenization using a tissue mincing device.

The comet assay is gaining popularity as a means to assess DNA damage in cultured cells and tissues, particularly following exposure to chemicals or other ...

Genetics

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Neuroscience

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Biology

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents is important for protecting public health. The in vivo alkaline comet assay, which detects DNA damage as strand breaks, is especially relevant for assessing the genotoxic hazards of xenobiotics, as its responses reflect the in vivo absorption, tissue distribution, metabolism and excretion (ADME) of chemicals, as well as DNA repair process. Compared to other in vivo DNA damage assays, the assay is rapid, sensitive, visual and inexpensive, and, by converting oxidative DNA damage into strand breaks using specific repair enzymes, the assay can measure oxidative DNA damage in an efficient and relatively artifact-free manner. Measurement of DNA damage with the comet assay can be performed using both acute and subchronic toxicology study designs, and by integrating the comet assay with other toxicological assessments, the assay addresses animal welfare requirements by making maximum use of animal resources. Another major advantage of the assays is that they only require a small amount of cells, and the cells do not have to be derived from proliferating cell populations. The assays also can be performed with a variety of human samples obtained from clinically or occupationally exposed individuals.

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

The alkaline comet assay measures DNA strand breaks in eukaryotic cells. By adding an Endonuclease III or human 8-oxoguanine-DNA-N-glycosylase digestion step, the assay can efficiently detect oxidative DNA damage. We describe methods for using these assays to detect DNA damage in rat liver.

Unrepaired DNA damage can lead to genetic instability, which in turn may enhance cancer development. Therefore, identifying potential DNA damaging agents ...

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell function, and therefore DNA damage may play a critical role in the development of, conceivably, all major human diseases, e.g., cancer, neurodegenerative and cardiovascular disease, and aging. Single-cell gel electrophoresis (i.e., the comet assay) is one of the most common and sensitive methods to study the formation and repair of a wide range of types of DNA damage (e.g., single- and double-strand breaks, alkali-labile sites, DNA-DNA crosslinks, and, in combination with certain repair enzymes, oxidized purines, and pyrimidines), in both in vitro and in vivo systems. However, the low sample throughput of the conventional assay and laborious sample workup are limiting factors to its widest possible application. With the "scoring" of comets increasingly automated, the limitation is now the ability to process significant numbers of comet slides. Here, a high-throughput (HTP) variant of the comet assay (HTP comet assay) has been developed, which significantly increases the number of samples analyzed, decreases assay run time, the number of individual slide manipulations, reagent requirements, and risk of physical damage to the gels. Furthermore, the footprint of the electrophoresis tank is significantly decreased due to the vertical orientation of the slides and integral cooling. Also reported here is a novel approach to chilling comet assay slides, which conveniently and efficiently facilitates the solidification of the comet gels. Here, the application of these devices to representative comet assay methods has been described. These simple innovations greatly support the use of the comet assay and its application to areas of study such as exposure biology, ecotoxicology, biomonitoring, toxicity screening/testing, together with understanding pathogenesis.

The comet assay is a popular means of detecting DNA damage. This study describes an approach to running slides in representative variants of the comet assay. This approach significantly increased the number of samples while decreasing assay run-time, the number of slide manipulations, and the risk of damage to gels.

Cells are continually exposed to agents arising from the internal and external environments, which may damage DNA. This damage can cause aberrant cell ...

Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy

Source: Lu, Y., et al. <a href="https://www.jove.com/v/56450">Evaluating In Vitro DNA Damage Using Comet Assay</a> J. Vis. Exp. (2017)
In this video, we demonstrate an alkaline single-cell electrophoresis assay to quantify both single- and double-strand DNA breaks in doxorubicin-treated cells. Alkaline conditions disrupt the hydrogen bonds between DNA base pairs, resulting in the complete unwinding and separation of DNA strands. This allows differential migration of undamaged and damaged DNA fragments in agarose gel upon electrophoresis, revealing a comet-shaped structure of slowly migrating undamaged DNA forming the circular comet head, and rapidly migrating damaged DNA fragments forming the elongated comet tail.

Elektroforeza w żelu alkalicznym z pojedynczymi komórkami: czuła technika ilościowego szacowania uszkodzeń DNA w poszczególnych komórkach rakowych po chemioterapii

Source: Lu, Y., et al. Evaluating In Vitro DNA Damage Using Comet Assay J. Vis. Exp. (2017)
In this video, we demonstrate an alkaline single-cell ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

Comet Assay for DNA Damage Detection in Cells

Source: Ji, Y., et al. <a href="https://app.jove.com/v/63559">A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage.</a> J. Vis. Exp. (2022).
The video demonstrates the comet assay for detecting DNA damage following exposure to harmful agents. Under alkaline conditions, the DNA unwinds and denatures, and upon electrophoresis, the undamaged and damaged DNA migrate differently in the agarose gel, forming a comet-shaped structure. The slowly migrating undamaged DNA forms the circular comet head, while the rapidly migrating damaged DNA fragments form the elongated comet tail.

Test komety do wykrywania uszkodzeń DNA w komórkach

Source: Ji, Y., et al. A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage. J. Vis. Exp. (2022).
The video demonstrates the comet ...

Evaluating In Vitro DNA Damage Using Comet Assay

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Przeglądaj więcej filmów

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

A Comprehensive Procedure to Evaluate the In Vitro Performance of the Putative Hemangioblastoma Neovascularization Using the Spheroid Sprouting Assay

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

Use of Frozen Tissue in the Comet Assay for the Evaluation of DNA Damage

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

In Vivo Alkaline Comet Assay and Enzyme-modified Alkaline Comet Assay for Measuring DNA Strand Breaks and Oxidative DNA Damage in Rat Liver

A High-Throughput Comet Assay Approach for Assessing Cellular DNA Damage

Elektroforeza w żelu alkalicznym z pojedynczymi komórkami: czuła technika ilościowego szacowania uszkodzeń DNA w poszczególnych komórkach rakowych po chemioterapii

Test komety do wykrywania uszkodzeń DNA w komórkach